1. Field of the Invention
This invention relates to synthetic soil extract substances comprised of phenolic polymers, and to compositions and methods for employing these synthetic phenolic polymers for preventing, reducing, treating, or eliminating influenza viral diseases.
2. Description of Related Art
Soil extract materials, particularly the classes of substances known collectively as xe2x80x9chumus,xe2x80x9d xe2x80x9chumics,xe2x80x9d xe2x80x9chumic acid(s),xe2x80x9d or xe2x80x9chumates,xe2x80x9d have been widely used in a number of applications for many years, as reviewed by F. J. Stevenson, Humus Chemistry. Genesis Composition Reactions; New York: Wiley, 1964; and, more recently, by A. Piccolo, Humic Substances in Terrestrial Ecosystems; New York: Elsevier, 1996.
Humic substances have long been known to exhibit anti-viral properties (H. Schultz, Dtsch. Tierarztl. Wochenschr. 1962, 69, 613; 1965, 72(13), 294-297; R. Klocking and M. Sprossig, Experientia 1972, 28(5), 607-608), particularly retroviruses (G. Sydow, V. Wunderlich, R. Klocking, and B. Helbig, Pharmazie 1986, 41(12), 865-868). Viral pathogens for which soil-extract materials have been shown to be effective include in particular Coxsackie virus A9 (Griggs-Baylor) (R. Klocking and M. Sprossig, Experientia 1972, 28(5), 607-608), herpes simplex virus type 1 (B. T. Rouse (Ed.), Herpes Simplex Virus; Berlin: Springer-Verlag, 1992; R. Klocking, K. D. Thiel, P. Wutzler, B. Helbig, and P. Drabke, Pharmazie 1978, 33(8), 539; F. Schiller, R. Klocking, P. Wutzler, and I. Farber, Dermatol. Monatsschr. 1979, 165(7), 505-509; B. Helbig, A. Sauerbrei, R. Klocking, P. Wutzler, N. Wicht, U. Wiedemann, and G. Herrmann, J. Med. Virol. 1987, 23(3), 303-309; R. Klocking and B. Helbig, in Humic Substances in the Aquatic and Terrestrial Environment; Berlin: Springer-Verlag, 1991; 407-412;) and type 2 (anon. Zentralbl. Bakteriol [Orig. A] 1976, 234(2), 159-169; K. D. Thiel, R. Klocking, H. Schweizer, and M. Sprossig, Zentralbl. Bakteriol [Orig. A] 1977, 239(3), 304-321; K. D. Thiel, B. Helbig, R. Klocking, P. Wutzler, M. Sprossig, and H. Schweizer, Pharmazie 1981, 36(1), 50-53; K. D. Thiel, B. Helbig, M. Sprossig, R. Klocking, and P. Wutzler, Acta Virol. 1983, 27(3), 200-208; K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782); human immunodeficiency virus (HIV) (M. Cushman, P. Wang, S. H. Chang, C. Wild, E. De Clercq, D. Schols, M. E. Goldman, and J. A. Bowen, J. Med. Chem. 1991, 34(1), 329-337; M. Cushman, S. Kanamathareddy, E. De Clercq, D. Schols, M. E. Goldman, and J. A. Bowen, J. Med. Chem. 1991, 34(1), 337-342; D. Schols, P. Wutzler, R. Klocking, B. Helbig, and E. De Clercq, J. Acquir. Immune Defic. Syndr. 1991, 4(7), 677-685; S. Loya, R. Tal, A. Hizi, S. Issacs, Y. Kashman, and Y. Loya, J. Nat. Prod. 1993, 56(12), 2120-2125; J. Schneider, R. Weis, C. Manner, B, Kary, A. Werner, B. J. Seubert, and U. N. Riede, Virology 1996, 218(2), 389-395; influenza virus type A (Krasnodar/101/59/H2N2) (R. Mentel, B. Helbig, R. Klocking, L. Dohner, and M. Sprossig, Biomed. Biochim. Acta 1983, 42(10), 1353-1356); and type B (J. Hils, A. May, M. Sperber, R. Klocking, B. Helbig, and M. Sprossig, Biomed. Biochim. Acta 1986, 45(9), 1173-1179); as well as other respiratory tract infectious agents (A. Jankowski, B. Nienartowicz, B. Polanska, and A. Lewandowicz-Uszyuska, Arch. Immunol. Ther. Exp. (Warsz) 1993, 41(1), 95-97).
The mechanisms whereby humic substances inhibit the cytopathicity of a number of viruses have been studied in some detail. It is thought that the materials prevent viral replication in part by sorbing onto the viral envelope protein (gp120 in the case of HIV), and thereby block the sorption of viral particles to cell surfaces: K. D. Thiel, R. Klocking, H. Schweizer, and M. Sprossig, Zentralbl. Bakteriol. [Orig. A] 1977, 239(3), 304-321; D. Schols, P. Wutzler, R. Klocking, B. Helbig, and E. De Clercq, J. Acquir. Immune Defic. Syndr. 1991, 4(7), 677-685; anon., Fortschr. Med. 1995, 113(7), 10; J. Schneider, R. Weis, C. Manner, B. Kary, A. Werner, B. J. Seubert, and U. N. Riede, Virology 1996, 218(2), 389-395. [Extracellular interception of pathogens by chemical agents that bind to them is a well-known means of immunological defense (D. M. Shankel, S. Kuo, C. Haines, and L. A. Mitscher, in Antimutagenesis and Anticarcinogenesis Mechanisms III; G. Bronzetti, H. Hayatsu, S. De Flora, M. D. Waters, and D. M. Shankel (Eds.); New York: Plenum, 1993; 65-74). Such materials might well be termed xe2x80x9cdespathogensxe2x80x9d, following the terminology proposed by T. Kada and K. Shimoi, Bioessays 1987, 7, 113-116, regarding xe2x80x9cdesmutagensxe2x80x9d.] It has also been found that naturally-occurring humic acid preparations can stimulate the production of cytokines, including interferon-gamma, interferon-alpha, and tumor necrosis factor-alpha (A. D. Inglot, J. Zielinksa-Jenczylik, and E. Piasecki, Arch. Immunol. Ther. Exp. (Warsz) 1993, 41(1), 73-80); as well as interferon-beta (Z. Blach-Olszewska, E. Zaczynksa, E. Broniarek, and A. D. Inglot, Arch. Immunol. Ther. Exp. (Warsz), 1993, 41(1), 81-85).
The toxicity of naturally-occurring humic acids is remarkably low (K. D. Thiel, B. Helbig, R. Klocking, P. Wutzler, M. Sprossig, and H. Schweizer, Pharmazie 1981, 36(1), 50-53; U. N. Riede, I. Jonas, B. Kim, U. H. Usener, W. Kreutz, and W. Schlickewey, Arch. Orthop. Trauma Surg. 1992, 111(5), 259-264; H. Czyzewska-Szafran, Z. Jastrzebski, D. Soltysiak-Pawluczak, M. Wutkiewicz, A. Jedrych, and M. Riemiszewska, Acta Pol. Pharm. 1993, 50(4-5), 373-377; H. L. Yang, F. J. Lu, S. L. Wung, and H. C. Chiu, Thromb. Haemost. 1994, 71(3), 325-330). [Cytotoxic effects of anti-viral substances, including humic acids, are usually evaluated via biological (viability and alterations of cell morphology) and biochemical testing methods (51Cr release), as described by K. D. Thiel, U. Eichhom, H. Schweizer, and R. Klocking, Arch. Toxicol. Suppl. 1980, 4, 428-430.] The cytotoxicity (CD50) of a naturally-occurring humic acid for human peripheral blood leukocytes (PBL) was found to be 1-9 milligrams per milliliter. In addition, J. Schneider, R. Weis, C. Manner, B. Kary, A. Werner, B. J. Seubert, and U. N. Riede, Virology 1996, 218(2), 389-395, reported that the cytotoxicity of a synthetic humic acid prepared from hydroquinone for MT-2 cells was approximately 600 micrograms per milliliter. It has also been found that medicaments prepared from humic acids isolated from naturally-occurring soil materials are neither carcinogenic (Syrian hamster embryo cell transformation test: J. Koziorowska and E. Anuszewska, Acta Pol. Pharm. 1994, 51(1), 101-102) nor mutagenic (T. Sato, Y. Ose, and H. Hagase, Mutat. Res. 1986, 162(2), 173-178; V. M. Sui, A. I. Kiung, and T. I. Veidebaum, Vopr. Kurortol. Fiozioter. Lech. Fiz. Kult. 1986, 2(3-4), 34-37; J. Koziorowska, B. Chlopkiewicz, and E. Anuszewska, Acta Pol. Pharm. 1993, 50(4-5), 379-382). Prenatal (S. Golbs, V. Fuchs, M. Kuhnert, and C. Polo, Arch. Exp. Veterinarmed. 1982, 36(2), 179-185) and embryotoxic and teratogenic effects (T. Juszkiewicz, M. Minta, B. Wlodarczyk, B. Biernacki, and J. Zmudzki, Acta Pol. Pharm. 1993, 50(4-5),383-388) are also not observed with humic preparations at daily dose levels from 5-50 milligrams per kilogram body weight. Topical preparations are tolerated even better (V. V. Soldatov and M. N. Cherepanova, Vopr. Kurortol. Fizioter. Lech. Fiz. Kult. 1970, 35(3), 256-259; H. Czyzewska-Szafran, Z. Jastrzebski, D. Soltysiak-Pawluczuk, M. Wutkiewicz, A. Jedrych, and M. Remiszewska, Acta Pol. Pharm. 1993, 50(4-5), 373-377) when applied dermally in aqueous solution in amounts as high as 10 percent weight-by-volume (K. Wiegleb, N. Lange, and M. Kuhnert, Dtsch. Tierarztl. Wochenschr. 1993, 100(10), 412-416).
Because humic substances are not chemically well-defined, the preparation of synthetic humic acids whose physicochemical properties mimic naturally-occurring materials is quite difficult, as pointed out by K. Murray and P. W. Linder, J. Soil Sci. 1983, 34, 511-523. Nevertheless, there have been several notable advances in this area. Broadly speaking, three general strategies have evolved. All depend upon starting with well-defined molecules of molecular weight on the order of hydroxybenzoic acid, and then causing the molecules to polymerize upon themselves to form larger molecules. The methods differ in the causation factor, which can be microbial, chemical, or enzymatic.
Humic acids of microbial origin have been described and discussed by M. Robert-Gero, C. Hardisson, L. Le Borgne, and G. Pignaud, Ann. Inst. Pasteur (Paris) 1966, 111(6), 750-767; and by M. Robert-Gero, C. Hardisson, L. Le Borgne, and G. Vidal, Ann. Inst. Pasteur (Paris) 1967, 113(6), 903-909.
The chemical synthesis of humic acids has been pioneered by R. Klocking, B. Helbig, and associates: R. Klocking, B. Helbig, and P. Drabke, Pharmazie 1977, 32, 297; R. Klocking, B. Helbig, K. D. Thiel, T. Blumohr, P. Wutzler, M. Sprossig, and F. Schiller, Pharmazie 1979, 34(5-6), 293-294; R. Mentel, B. Helbig, R. Klocking, L. Dohner and M. Sprossig, Biomed. Biochim. Acta 1983, 42(10), 1353-1356; H. P. Klocking, R. Klocking, and B. Helbig, Farmakol. Toksikol. 1984, 47(1), 93-95; K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782; J. Hils, A. May, M. Sperber, R. Klocking, B. Helbig, and M. Sprossig, Biomed. Biochim. Acta 1986, 45(9), 1173-1179; B. Helbig, A. Sauerbrei, R. Klocking, P. Wutzler, N. Wicht, U. Wiedemann, and G. Herrmann, J. Med. Virol. 1987, 23(3), 303-309; K. I. Hanninen, R. Klocking, and B. Helbig, Sci. Total Environ. 1987, 62, 201-210; R. Klocking and B. Helbig, in Humic Substances in the Aquatic and Terrestrial Environment; New York: Springer-Verlag, 1989; 407-412; C. Schewe, R. Klocking, B. Helbig, and T. Schewe, Biomed. Biochim. Acta 1991, 50(3), 299-305; D. Schols, P. Wutzler, R. Klocking, B. Helbig, and E. De Clercq, J. Acquir. Immune Defic. Syndr. 1991, 4(7), 677-685. Typically, 10 millimoles of the starting small-molecule phenolic compound is dissolved in distilled water, the pH is adjusted to 8.5 with aqueous sodium hydroxide (NaOH), and then 2-5 millimoles of sodium periodate (NaIO4) is added. The solution is warmed at 50xc2x0 C. for 30 minutes, and is then allowed to stand overnight. The resultant humic acid-like polymeric products are isolated by precipitation with lead(II) nitrate [Pb(NO3)2]. The precipitated polymers are redissolved in aqueous sodium hydroxide (pH 8.5) and heated with 8-hydroxyquinoline for 30 minutes at 100xc2x0 C. The precipitate formed is lead(II) chelate, which is removed by filtration. Residual 8-hydroxyquinoline is extracted with chloroform, and the desired polymeric material is then precipitated from the aqueous solution by the addition of various combinations of acetic acid, ethyl acetate, and ethanol. Starting compounds that have been used for the synthesis of humic-like materials include 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one (aurin), 4-[bis(3-carboxy-4-hydroxyphenyl)methylene]-2-carboxy-2,5-cyclohexa-dien-2-one (aurintricarboxylic acid), 3-(3,4-dihydroxyphenyl)propenoic acid (caffieic acid), 1,2-dihydroxybenzene (catechol), 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid 3-(3,4-dihydroxyphenyl)propenoate (chlorogenic acid), 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid), 1-(3,4-dihydroxyphenyl)-2-(N-methylamino)ethanol (epinephrine), 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid (ferulic acid), 3,4-5-trihydroxybenzoic acid (gallic acid), 2,5-dihydroxybenzoic acid (gentisic acid), 2,5-dihydroxyphenylacetic acid (homogentisic acid), 3-(3,4-dihydroxyphenyl)propionic acid (hydrocaffeic acid), 1,4-dihydroxybenzene (hydroquinone), 2,3-dihydroxytoluene (3-methylcatechol), 3,4-dihydroxytoluene (4-methylcatechol), 2,5-dihydroxytoluene (2-methylhydroquinone), 4,4xe2x80x2-(2,3-dimethyltetramethylene)-di-(1,2-dihydroxybenzene) (nordihydroguaiaretic acid), 1-(3,4-dihydroxyphenyl)-2-aminoethanol (norepineph-rine), 3,4-dihydroxybenzoic acid (protocatechuic acid), 1,2,3-trihydroxybenzene (pyrogallol), 1,3-dihydroxybenzene (resorcinol), and 4-hydroxy-3-methoxybenzoic acid (vanillic acid). Other notable efforts on the chemical synthesis of humic-like substances include the studies by De Clercq and colleagues on aurintricarboxylic acid, its derivatives, and related compounds: M. Cushman, P. Wang, S. H. Chang, C. Wild, E. De Clercq, D. Schols, M. E. Goldman, and J. A. Bowen, J. Med. Chem. 1991, 34(1), 329-337; M. Cushman, S. Kanamathareddy, E. De Clercq, D. Schols, M. E. Goldman, and J. A. Bowen, J. Med. Chem. 1991, 34(1), 337-342. Related efforts have also been reported by M. Robert-Gero, C. Hardisson, L. Le Borgne, and G. Vidal, Ann. Inst. Pasteur (Paris) 1967, 113(6), 903-909; M. Jakubiec, E. Miszczak, and J. Szczerkowska, Acta Microbiol. Pol. [B] 1971, 3(1), 63-66; R. Ansorg and W. Rochus, Arzneimittelforschung 1978, 28(12), 2195-2198; J. Pommery, M. Imbenotte, A. F. Urien, D. Marzin, and F. Erb, Mutat. Res. 1989, 223(2), 183-189; F. J. Lu and Y. S. Lee, Sci. Total Environ. 1992, 114, 135-139; K. Wiegleb, N. Lange, and M. Kuhnert, DTW Dtsch. Tierarztl. Wochenschr. 1993, 100(10), 412-416; H. L. Yang, F. J. Lu, S. L. Wung, and H. C. Chiu, Thromb. Haemost. 1994, 71(3), 325-330; W. Seffner, F. Schiller, R. Heinze, and R. Breng, Exp. Toxicol. Pathol. 1995, 47(1), 63-70; and J. Schneider, R. Weis, C. Manner, B. Kary, A. Werner, B. J. Seubert, and U. N. Riede, Virology 1996, 218(2), 389-395.
The enzymatic catalytic synthesis of humic acids dates to about 1961 with the work by R. E. Hampton and R. W. Fulton, Virology 1961, 13, 44-52 (see also R. E. Hampton, Phytophathology 1970, 60, 1677-1681), who found that enzymatically oxidized phenols inactivate phytopathogenic (i.e., plant-related) viruses. Typically o-diphenol oxidase has been employed for the enzymatic synthesis of humic-like materials: anon. Zentralbl. Bakteriol. [Orig A] 1976, 234(2), 159-169; R. Klocking, B. Helbig, and P. Drabke, Pharmazie 1977, 32(5), 297; K. D. Thiel, B. Helbig, R. Klocking, P. Wutzler, M. Sprossig, and H. Schweizer, Pharmazie 1981, 36(1), 50-53; K. D. Thiel, B. Helbig, M. Sprossig, R. Klocking, and P. Wutzler, Acta Virol. 1983, 27(3), 200-208; K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782; and G. Sydow, V. Wunderlich, R. Klocking, and B. Helbig, Pharmazie 1986, 41(12), 865-868.
A direct comparison of humic acids synthesized enzymatically and nonenzymatically from caffeic and hydrocaffeic acids has shown that the two synthetic routes produce materials that differ somewhat in their efficacy for the suppression of herpes (hominis) types 1 and 2 viruses: K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782.
PCT application WO 00/16785 (Mar. 20, 2000) from Dekker and Medlen discloses the use of humic acid or its salts, esters, or derivatives thereof, all prepared as described in U.S. Pat. Nos. 4,912,256 and 5,004,831 from coal extracts, in stimulating lymphocytes in a human, animal, or bird. This allows for the treatment of viral and bacterial infections, and more particularly HIV infections, cancer, and opportunistic diseases. Oxihumic acids, salts, esters, or derivatives thereof are preferred. Administration is preferably oral. Some example pharmacological data presented include the antiviral activity of oxihumates against HIV-1 in vitro and clinical trials of oral oxihumate in HIV-infected patients.
PCT application WO 00/16786 (Mar. 30, 2000) from Dekker and Medlen discloses the use of pharmaceutical compositions comprising an oxihumic acid or its salts, esters, or derivatives thereof, all prepared as described in U.S. Pat. Nos. 4,912,256 and 5,004,831 from coal extracts, as active ingredients. Compositions are preferably administered orally for stimulating lymphocytes in a human, animal, or bird. They may be used in treating viral and bacterial infections, HIV infections, opportunistic diseases, inflammation, pain and fever, cancer growth, and diseases associated with viral infection and a depressed immune system. A number of pharmacological examples are given, including interleukin 10 production by oxihumate-reated lymphocytes, increased antibody production against Newcastle disease in chickens treated with oxihumate, TNF production by oxihumate-treated lymphocytes, and antiviral activity of oxihumate against HSV-1 and coxsackie virus type 1 in vitro.
The diversity of physicochemical characteristics as well as wide variation in the biological activity and toxicity of humics extracted or otherwise derived from natural soils has been well documented. This diversity and variation is due to variations in factors such as the source of the soil, the method(s) of extraction and/or isolation, and the technique(s) employed to treat the extract once it has been separated and isolated from crude soil. The consequence of irreproducibility of the properties of substances extracted from natural soil is that the commercial value of such materials is minimized. In addition, they are rendered unsuitable as medicaments. Also, while a number of laboratory-scale processes have already been described that address various aspects of the isolation, synthesis, and/or preparation of humic substances or similar materials, there are no reports of preparing and isolating such purely synthetic humic acids or similar materials by methods that are suitable for scaleup directly to industrial levels, that provide economically acceptable yields, and that optimize the preparation procedures from the standpoint of medicament safety and efficacy. All of the known synthetic methods utilize potentially toxic precipitation methods [lead(II) nitrate precipitation] followed by complex isolation procedures, potentially mutagenic compound-producing hydrochloric acid precipitation or lengthy synthetic steps as long as 10 days.
There is a need to devise simple synthetic procedures that yield inexpensive, safe materials whose physicochemical attributes are reproducible, and that at least simulate those of typical commercially-available soil extracts.
One embodiment is a method for preventing and/or treating influenza virus infection in a mammal which comprises administering an effective amount of a synthetic phenolic polymeric material which is prepared by:
a) dissolving in an aqueous solution at least one starting organic compound comprising at least one hydroxyl group and at least one carbonyl group or at least two hydroxyl groups on an aromatic structure;
b) adjusting the pH of the aqueous solution resulting from step a) to between about 8 and 11;
c) adding an alkaline periodate salt or alkaline-earth periodate salt to the aqueous solution resulting from step b);
d) maintaining the temperature of the solution from step c) between about 20xc2x0 C. and 100xc2x0 C. for a period of at least about 30 minutes;
e) adding at least one water soluble compound or salt selected from the group consisting of boric acid, borate salts, alkaline earth salts, transition metal salts, alkaline sulfides, alkaline earth sulfides, or transition metal sulfides to the aqueous solution resulting from step d);
f) allowing the aqueous solution from step e) to stand with or without stirring at about 20xc2x0 C. to 100xc2x0 C. for at least about 2 hours; and
g) removing molecules from the solution resulting from step f) below about 500 to about 10,000 daltons.
In another aspect, the method of preparation of the synthetic phenolic polymeric material further comprises a step, following the step of removing molecules from the solution below about 500 daltons to 10,000 daltons, of concentrating the solution.
In another aspect, the method of preparation of the synthetic phenolic polymeric material further comprises a step, following the step of removing molecules from the solution below about 500 dalton to 10,000 daltons, of removing water from the solution.
In another aspect, the influenza virus infection is effected by a virus, preferably influenza A or influenza B.
In another aspect, the administering of a synthetic phenolic polymeric material is along with an effective amount of an antiviral composition.
In another aspect, the administering of a synthetic phenolic polymeric material can be achieved systemically or topically.
One embodiment is a method for inhibiting influenza viral attachment to host cells in a mammal which comprises administering an effective amount of a synthetic phenolic polymeric material which is prepared by:
a) dissolving in an aqueous solution at least one starting organic compound comprising at least one hydroxyl group and at least one carbonyl group or at least two hydroxyl groups on an aromatic structure;
b) adjusting the pH of the aqueous solution resulting from step a) to between about 8 and 11;
c) adding an alkaline periodate salt or alkaline-earth periodate salt to the aqueous solution resulting from step b);
d) maintaining the temperature of the solution from step c) between about 20xc2x0 C. and 100xc2x0 C. for a period of at least about 30 minutes;
e) adding at least one water soluble compound or salt selected from the group consisting of boric acid, borate salts, alkaline earth salts, transition metal salts, alkaline sulfides, alkaline earth sulfides, or transition metal sulfides to the aqueous solution resulting from step d);
f) allowing the aqueous solution from step e) to stand with or without stirring at about 20xc2x0 C. to 100xc2x0 C. for at least about 2 hours; and
g) removing molecules from the solution resulting from step f) below about 500 to about 10,000 daltons.
In another aspect, the method of preparation of the synthetic phenolic polymeric material further comprises a step, following the step of removing molecules from the solution below about 500 daltons to 10,000 daltons, of concentrating the solution.
In another aspect, the method of preparation of the synthetic phenolic polymeric material further comprises a step, following the step of removing molecules from the solution below about 500 dalton to 10,000 daltons, of removing water from the solution.